BETA-ADRENERGIC AND CHOLINERGIC MODULATION OF INWARD RECTIFIER K+ CHANNEL FUNCTION AND PHOSPHORYLATION IN GUINEA-PIG VENTRICLE

1. To clarify the nature of the inhibition of whole-cell inwardly rectifying K+ current (I-k1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (i(k1)) carried by inwardly rectifying K+ channels in cell-attached and excised inside-out patches obtained from guinea-pig ventricular myocytes. 2. Bath application of Iso suppressed i(K1) channel activity in cell-attached patches. This was inhibited by propranolol. Bath-applied forskolin or dibutyryl cAMP mimicked the effect of bath-applied Iso. 3. Exposure of the cytosolic face of inside-out patches to purified catalytic subunit of the cAMP-dependent protein kinase (PKA) also suppressed i(K1) channel activity, mimicking the effect of bath-applied Iso on i(K1) recorded from cell-attached patches. 4. When applied directly to cell-attached patches via the patch pipette solution, ACh antagonized Iso-induced (1 mu M applied via the bath) suppression of i(K1) channels. In contrast, bath-applied ACh(10 mu M) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on i(K1) channels in cell-attached patches but had no detectable effect when 1 mu M or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso-induced suppression of i(K1) channels. When inside-out patches were used, bath-applied preactivated exogenous inhibitory G protein subunit, G(1 alpha) antagonized the suppression of i(K1) channels induced by bath-applied catalytic subunit of PKA (PKA-CX), suggesting that a PTX-sensitive G(i alpha) mediates ACh-induced antagonism of Iso-induced suppression of i(K1). 6. Neither GTP gamma S nor G(i alpha) antagonized the suppression of i(K1) produced by bath-applied PKA-CX in inside-out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated i(K1) channels suppressed by PXA-CS. 7. Findings in guinea-pig ventricular myocytes suggest that i(K1) can be suppressed by a PKA-mediated phosphorylation of the i(K1) channel occurring in response to Iso-induced beta-adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane-delimited pathways. The membrane-delimited pathway appears to involve M(2)-cholinergic receptors, their associated G protein, G(1), and a protein phosphatase, all located in the sarcolemma in close proximity to the involved i(K1) channels.